The present invention, in some embodiments thereof, relates to methods of inducing proliferation of cardiomyocytes and methods of treating heart diseases.
Heart disease and in particular myocardial infarction (MI), is the leading cause of death in the world. The severity of heart disease is due to the post-mitotic nature of mammalian adult cardiac muscle cells—the cardiomyocytes (CMs) {Bergmann, 2009 #9; Senyo, 2013 #86} and their limited capacity to replenish damaged tissue {Poss, 2007 #30; Ausoni, 2009 #17}. In contrast, extensive CM proliferation and subsequently robust cardiac regeneration occurs in lower vertebrates such as newt {Ausoni, 2009 #17} and zebrafish {Poss, 2007 #30; Jopling, 2010 #68; Ausoni, 2009 #17}. Similarly, neonatal murine CM turnover is sufficient to repair damaged myocardium following injury; however this ability is greatly diminished during the first week after birth {Porrello, 2011 #11; Porrello, 2012 #38}. During this time, there is a transition in CM ploidy from mono to bi-nucleation, concurrent with a switch from hyperplasia (increase in cell number) to hypertrophy (increase in cell size) {Li, 1996 #61; Soonpaa, 1998 #89}. Induced cardiac injury in mice at the day of birth results in nearly complete regeneration however this capacity is diminished by day 7. At this time point fibrotic scar dominates the replenishment of muscle tissue through CM proliferation {Porrello, 2011 #11} therefore leading to impaired cardiac function {Weisman, 1988 #90}. Many studies focus on the proliferation of endogenous CMs in order to contribute to heart regeneration. Recently, it was shown that adult CMs can re-enter the cell cycle and proliferate by modulating several pathways such as: FGF1 accompanied with P38 inhibition {Engel, 2006 #127}, extracellular Periostin {Kuhn, 2007 #28}, NRG1 via Erbb2 {D'Uva, 2015 #99; Bersell, 2009 #128} Hippo inhibition {Heallen, 2013 #100} and inhibition of the cell cycle regulator Meisl {Mahmoud, 2013 #80}.
Heart pathologies, primarily MI, are often accompanied by ECM remodeling, mainly deposition of a rigid scar which reduces heart function {Weisman, 1988 #90; Baum, 2011 #12; Bayomy, 2012 #3}. Alterations in ECM structure following injury are attributed to activity of matrix metalloproteases (MMPs) {Phatharajaree, 2007 #92}, mainly the gelatinase family, MMP2 and MMP9 {DeCoux, 2014 #91}. Deletion of either MMP2 {Hayashidani, 2003 #94} or MMP9 {Ducharme, 2000 #93} following MI attenuated ECM remodeling and improved overall heart function. Despite the adverse effects of ECM remodeling following cardiac injury, ECM plays an integral role in cellular migration {Ridley, 2003 #95; Berk, 2007 #97}, differentiation{Shamis, 2011 #18; Streuli, 1999 #96} and proliferation {Berk, 2007 #97} of any cell type.
Through utilization of ECM decellularization and acid solubilization of fetal, neonatal and adult cardiac ECM, cardiac ECM was shown to significantly contribute to the regulation of CM proliferation {Williams, 2014 #84}. Seeded on neonatal cells, ECM derived from fetal and neonatal ages displayed higher proliferation levels compared to adult derived ECM {Williams, 2014 #84}. Although manipulation of CM intrinsic factors was shown to expand the proliferative capacity of the mammalian heart {D'Uva, 2015 #99; Mahmoud, 2013 #80; Heallen, 2013 #100}, the roles of the extracellular environment or its components in cardiac regeneration remain unclear.
Agrin is an extracellular heparan sulfate proteoglycan (HSPG) with a core protein size of 210 kDa {Williams. 2008 #71}. The neural form of Agrin (n-Agrin) has been extensively researched due to its involvement in the aggregation of acetylcholine receptors (AChRs) via the muscle specific kinase (MuSK)-Lrp4 receptor complex {Burden, 2013 #76}. Elevated expression of non-neuronal Agrin has been correlated with several types of carcinoma {Theocharis, 2010 #102} and more recently has been implicated in the progression of hepatocellular carcinoma (HCC) by controlling motility and proliferation of cells through interaction with Lrp4 {Chakraborty, 2015 #101}. Additionally, a small fragment of Agrin (the c-terminal 22 kDa peptide, CAF22) has been shown to bind and inhibit Na+ K+ channels that modulate CM beating {Hilgenberg, 2009 #103}, similarly to Digoxin, a drug commonly taken after various cardiac episodes {Hilgenberg, 2009 #103; Schwinger, 2003 #104}. A recent study focused on the interaction of Agrin with the dystroglycan complex as a key component in processes of innate immunity, and is required for monocyte and macrophage differentiation through interaction with Grb2 and subsequent ERK activation {Mazzon, 2012 #73}.
Dystroglycan is comprised of two units (α and β) {Henry, 1996 #105} and acts as a transmembrane bridge connecting ECM components (such as Agrin, Laminin and Perlecan) with the muscle cell inner myoskeleton by interacting with Dystrophin and its associated complex {Henry, 1996 #105; Davies, 2006 #106; Ervasti, 1990 #108}. Interruption of Dystrophin complex is the leading cause for various muscular dystrophies including Duchenne muscular dystrophy {Davies, 2006 #106; Campbell, 1989 #107; Ervasti, 1990 #108}. Mice lacking Dystrophin (Mdx), present elevated CM turnover in non-ischemic cardiomyopathy model {Richardson, 2015 #110}; In contrast, a recent study that employed post-natal day 1 heart resection on Mdx mice revealed impaired regenerative response and elevated fibrosis relative to wildtype mice {Morikawa, 2015 #109}. Nonetheless, the role of Agrin, Dystroglycan and their downstream elements has never been studied in the context of cardiac regeneration.
Additional background art includes:
U.S. Application Number 20070014871
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U.S. Application Number 20110104120
U.S. Application Number 20070014733